HPV-16 E6、E7基因慢病毒真核表达载体的构建及鉴定

Construction and identification of eukaryotic vector of HPV-16 E6,E7 genes

目的构建人乳头瘤病毒(HPV)E6、E7基因慢病毒真核表达载体。方法以Si Ha宫颈癌细胞总RNA为模板,利用RT-PCR技术扩增出E6、E7目的片段;选用病毒载体p Lenti6/V5 Directional TOPO Cloning Kit系统构建E6、E7基因慢病毒真核表达载体;分别使用构建好的E6、E7载体转染病毒包装细胞,获得含病毒的上清液,并测定上清液中慢病毒的滴度。结果质粒测序所得序列与人乳头瘤病毒E6、E7基因序列完全吻合,载体构建成功;慢病毒滴度测定结果显示E6样品的慢病毒滴度为1.5×108TU/m L,E7样品的慢病毒滴度为2×108TU/m L,该滴度能够满足后续研究所需。结论 HPV-16 E6、E7基因慢病毒真核表达载体构建成功,可以为进一步研究人乳头瘤病毒E6、E7基因致病机理奠定物质基础。

Objective To construct the eukaryotic expression vector for human papillomavirus （HPV） E6 and E7 genes. Methods We amplified the full-length HPV-16 E6 and E7 genes from the total RNA of the SiHa cell line by RT-PCR technique and sub-cloned the gene into pLenti6/V5 vector, which was identified and confirmed by DNA sequencing. The construction of E6 and E7 transfected viruses was used to package cells. Then we harvested viral supematant and determined the titer. Results The plasmid sequencing was completely consistent with E6, E7 gene sequence, which showed that the vector was successfully construc- ted. The titer of E6 was 1.5× 108 TU/mL, and 2× 108 TU/mL of E7, satisfying the requirements of the subsequent researches. Conclusion The successful construction of HPV-16 E6 and E7 gene eukaryotic ex- pression vector could lay the material foundation for furthering study of the pathogenesis of HPV E6 and E7 genes.